The ability of the intestinal cell to export triacylglycerol (TG) is a physiologically regulatable function. The intracellular site where this occurs is unknown, although available evidence suggests that the step between the endoplasmic reticulum (ER) and the Golgi is the most likely. We studied this process in rat enterocytes that were isolated from the proximal intestine. A novel system was developed in which [3H]TG was transported from ER to the Golgi. This process was time, ATP, temperature, and cytosol dependent. The cytosolic factor(s) was heat and trypsin sensitive. TG transport was directly proportional to the amount of added nonradiolabeled acceptor Golgi. The rate of TG transported to the Golgi was the fastest in cells isolated from rats that had been intraduodenally infused in vivo with glyceryltrioleate (TO) plus phosphatidylcholine and slowest in cells isolated from bile-fistulated rats infused with TO in vivo compared with cells from in vivo TO-infused, bile duct intact rats, mimicking the relative transport rates seen in vivo. TG transport in vitro could not be quenched by adding TG emulsions, chylomicrons, liposomes, or guanosine 5'-O-(3-thiotriphosphate). Cytosol from the liver and kidney supported TG transport, but the Golgi from liver or kidney did not accept TG from intestinal ER. We conclude that an intestinally specific, active transport mechanism transports TG from the ER to the Golgi and that this might be a regulatory step in TG export from the intestinal cell.